The Formation of Amyloid-Like Fibrils of α-Chymotrypsin in Different Aqueous Organic Solvents
2012
The formation of amyloid-like fibrils of α-chymotrypsin was studied in aqueous ethanol, methanol, tertbutanol,
dimethylformamide and acetonitrile. Thioflavin T (ThT), Congo red (CR) and 1-anilino-8-naphthalenesulfonic
acid (ANS) binding, turbidity, intrinsic fluorescence and far-UV circular dichroism measurements were employed to characterize
the amyloid fibril formation. The greatest extent of fibril formation after incubation for 24 h at pH 7.0 and at 24
oC was in ethanol at 55%, in methanol and dimethylformamide (DMF) at 60-70% and in tert-butanol at 60-80%. The ANS
binding and intrinsic fluorescence results showed that the hydrophobic residues are more solvent-exposed in the aggregated
form of α-chymotrypsin. The ThT, CR binding and far-UV CD measurements indicated that the formation of the
cross-β structure of α-chymotrypsin depends on the polarity of the organic solvent. To determine the role of surface
charges in the aggregation, chemically modified forms of α-chymotrypsin were prepared. The citraconylated and succinylated
enzymes exhibited a higher and the enzyme forms modified with aliphatic aldehydes a lower propensity for aggregation.
These results suggest the important role of surface charges in the aggregation of α-chymotrypsin.
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